Immobilized carboxypeptidase G1 in methotrexate removal.

Carboxypeptidase G1, and enzyme capable of cleaving the glutamate moiety from a variety of folate analogs, has been immobilized on nylon tubes and hollow fibers for use in extracorporeal enzyme reactors for methotrexate (MTX) removal from blood. The stability and reactor parameters of the system have been investigated with the use of single tubes and a multitubular arrangement. The results are used to predict their MTX-removing capacity at flow rates and MTX concentrations of clinical interest. In vivo experiments with dogs demonstrate the potential applications of such extracorporeal shunts in "rescue" after administration of large doses of MTX. For dogs a carboxypeptidase G1 reactor with a clearance value of about 150 ml/min would be required in such application.

Enhancement of antitumor activity of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine and Baker's antifol (triazinate) with carboxypeptidase G1.

Carboxypeptidase G1 (CPDG1), an enzyme that degrades folates but not the nonclassical folate antagonists triazinate (TZT, Baker's antifol) and 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP), enhanced the antineoplastic activity of these antifolates. In 6-day cell culture experiments with Walker 256 carcinosarcoma, CPDG1 at levels up to 0.54 unit/ml showed very little inhibitory effect on growth. In the presence of 10(-7) M DDMP, the grown of Walker 256 cells was similar to that of controls, but in combination with CPDG1 (0.1 unit/ml) 80% growth inhibition was observed. TZT at a concentration of 10(-8) M did not affect the growth of Walker 256 cells. The combination of 10(-8) M TZT with CPDG1 (0.1 unit/ml), however, strongly inhibited cell growth. In experiments with rats bearing Walker 256 carcinosarcoma, the administration of CPDG1 (800 units/kg/day) from Day 1 to Day 6 resulted in no significant increase in life span. Administration of TZT in doses up to 0.05 mg/kg on Day 1 or that of DDMP up to 15 mg/kg on Days 1, 3, and 5 had no antitumor effects as measured by survival of the animals. However, CPDG1 (800 units/kg/day) from Day 1 to Day 6 in combination with TZT (0.05 mg/kg on Day 1) or DDMP (15 mg/kg on Days 1, 3, and 5) resulted in increases of 50 and 30%, respectively, in the survival of the tumor-bearing animals. These results demonstrate that CPDG1 enhances the antitumor effects of TZT or DDMP.

Occurrence and significance of D-methotrexate as a contaminant of commercial methotrexate.

Methotrexate from various commercial sources has been found to contain 0.5 to 48% (w/w) of the enantiomer D-methotrexate. The two methotrexate enantiomers were separated by using chiral high-performance liquid chromatography with an octadecyl silica column and a mobile phase containing L-proline and cupric nitrate. For the assay of D-methotrexate impurity in commercial methotrexate, L-methotrexate was hydrolyzed with carboxypeptidase G1, and the remaining D-methotrexate was quantitated by high-performance liquid chromatography. The biological effects of D-methotrexate were investigated and compared to that of L-methotrexate. D-Methotrexate was found to be a good inhibitor of dihydrofolate reductase from both murine and human tumor cells, but was a poor inhibitor of L1210 and CCRF-CEM cell growth. In animal experiments with dogs and mice, D-methotrexate was rapidly absorbed from the intestine and excreted by the kidneys.

Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents.

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C18 micropellicular and Vydac C4 and C18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C4 and the Hy-Tech C18 column, respectively.

Anisotropic membranes with carboxypeptidase G1.

Anisotropic polysulfone membranes were prepared with carboxypeptidase G1 embedded in the polymer structure. The enzymatically active flat and hollow-fiber membranes were obtained by precipitating the polymer from solution in an organic mixture in which an aqueous solution of the enzyme had been dispersed. The process has been found to be particularly suitable for the immobilization of enzymes in anisotropic hollow fibers that exhibited no detectable enzyme leakage upon perfusion. The pH profiles measured with the enzyme in free solution and in the embedded form were similar. Kinetic parameters of multitubular enzyme reactors were investigated by measuring the rate of hydrolysis of glutamate from folic acid or methotrexate at different flow rates and substrate concentrations. The relatively slow mass transfer in such reactors was found to affect strongly the observed kinetics. The results of in vitro experiments with 5000 fiber reactors suggest that hollow fiber cartridges prepared with such membranes have clinical potential for the extracorporeal removal of methotrexate from blood.

Micropellicular stationary phases for rapid protein analysis by high-performance liquid chromatography.

The high separating speed, efficiency and operational stability of various micropellicular stationary phases are demonstrated in the high-performance liquid chromatography (HPLC) of biopolymers. The micropellicular sorbents were prepared from 2-microns fluid-impervious silica microspheres as the support, with a thin layer of different retentive materials at the surface. These include a molecular fur of octyl or stearyl chains for reversed-phase chromatography as well as a hydrophilic layer with amino groups and polyethyleneglycol chains for anion-exchange and hydrophobic interaction chromatography, respectively. The use of appropriate micropellicular stationary phases for protein separation by metal-interaction and affinity chromatography is also illustrated. In most cases, operation at elevated column temperature was found to be preferable for rapid separations. Preliminary results show that the stability of micropellicular columns compares very favorably with that of columns conventionally used in HPLC and that they are easy to maintain.

Rapid peptide mapping by high-performance liquid chromatography.

Short columns, packed with pellicular sorbents made of 2-micron fluid-impervious silica microspheres, were used at elevated column temperatures for rapid peptide mapping by high-performance liquid chromatography (HPLC). Enzymic digests of various proteins were chromatographed by gradient elution. In many cases the time of analysis was 10 min or less. In order to increase the retention particularly, that of short, polar peptides under such conditions, 1 mM octyl sodium sulfate or 5 mM hexyl sodium sulfate were added to the starting eluent. The length of the 4.6 mm I.D. columns was 30 or 75 mm, the sample load was in the range of 10-1000 pmoles. Highest analytical sensitivity was obtained at a flow-rate of 0.5 ml/min and room temperature, whereas for rapid analysis flow-rates of up to 2 ml/min were used at 80 degrees C. This temperature allowed the use of relatively high flow velocities of the mobile phase without significant loss in efficiency. The method was highly reproducible, as shown by the results obtained by automated analysis of cyanogen bromide fragments of lysozyme at high speed. The quality of the rapid peptide maps compares favorably with that of maps obtained by standard reversed-phase HPLC methods, which require much longer analysis times.

Rapid displacement chromatography of melittin on micropellicular octadecyl-silica.

Rapid high-performance liquid chromatographic analysis and displacement purification of melittin and its variants were carried out by reversed-phase chromatography. High speed of separation was achieved by the use of columns packed with a micropellicular stationary phase consisting of a thin C18 hydrocarbonaceous layer on the surface of 2-microns fluid-impervious silica microspheres at elevated temperature. In the case of melittin from bee venom or its synthetic variants the plots of the logarithmic retention factor against acetonitrile concentration in the eluent were straight lines whereas the van't Hoff plots in the temperature range from 20 to 80 degrees C were non-linear. Purification of melittins by displacement was carried out with benzyldimethylhexadecyl ammonium chloride as the displacer. In a 20-min displacement run at 40 degrees C about 5 mg of highly pure melittin were isolated from 10 mg of synthetic mixture by using a 105 x 4.6 mm column. The results demonstrate that columns packed with micropellicular sorbents not only facilitate rapid high-performance liquid chromatographic analysis but are also suitable for fast peptide purification with high recovery.

Rapid high-performance affinity chromatography on micropellicular sorbents.

Short columns (30 x 4.6 mm I.D.), packed with 2-micron fluid-impervious silica microspheres with surface-bound Protein A or a lectin were used for fast separation and quantitation of immunoglobulins and glycoproteins by biospecific interaction chromatography. With stepwise elution, the total analysis time including column reequilibration did not exceed 3 min. In the assay of IgG with a stepwise change in pH best results were obtained with citrate buffer, which facilitated not only fast but also very sensitive analysis. The calibration curve was linear in the range 0.5-40 micrograms of human IgG. By using morpholinoethanesulfonic acid-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-acetic acid buffer with a linear decrease in pH from 6.0 to 4.0 and an increase in magnesium chloride concentration to 200 mM for elution, the subclasses of human IgG were separated at 40 degrees C above pH 4.0 in 3 min. Micropellicular concanavalin A and wheat germ agglutinin were used for rapid affinity chromatography of horseradish peroxidase and fetuin, respectively. The results suggest that micropellicular affinity sorbents afford fast and sensitive high-performance liquid chromatographic analysis by biospecific interaction chromatography. Although developed primarily for rapid analysis, the micropellicular Protein A exhibited unexpectedly high adsorption capacity (e.g., 4.5 mg human IgG per ml of wet bed volume). This suggests that such columns could be employed in preparative protein chromatography as well.

Multitubular reactors with immobilized L-phenylalanine ammonia-lyase for use in extracorporeal shunts.

Hollow fiber cartridges of improved design have been loaded with L-phenylalanine ammonia-lyase [EC 4.3.1.5.] from Rhodotorula glutinis in order to prepare multitubular enzyme reactors for removal of L-phenylalanine from blood. The kinetics of the enzyme in free solution was investigated and the results showed a behavior similar to that reported for L-phenylalanine ammonia-lyase from other sources. On the other hand kinetic data with the reactors were obtained in a recirculating system by measuring the increase in trans-cinnamate concentration in the perfusate. The effect of changing the enzyme load in the hollow fiber cartridge, the length of the cartridge, the substrate concentration, pH as well as the flow rate and the temperature have been investigated. The results showed that the overall kinetics of the enzymic reaction in the multitubular reactor is not the same as that in free solution and the difference can be attributed to diffusion resistances in the hollow fiber system.

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani.

1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate.

Determination of aromatic amino acids by ion-pair reversed-phase liquid chromatography in human sera from healthy and phenylketonuric individuals.

A simple chromatographic procedure is described for simultaneous analysis of aromatic amino acids in serum samples obtained from normal individuals and from phenylketonuric patients. Quantitative measurement of phenylalanine, tyrosine, histidine and tryptophan is made in samples as small as 10 microliter after deproteinization with trichloroacetic acid. With phenoxyacetic acid as the internal standard, samples are analyzed by reversed-phase chromatography isocratically by an ion-pairing agent in the eluent, and the amino acids are detected with the UV detector at 210nm. Total analysis time was about 30 minutes. Using this method in serum samples from phenylketonuric patients phenylalanine was increased (as expected), and histidine was decreased, at a statistically significant level.

A biophysical study of integral membrane protein folding.

In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.

In vivo safety of hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase in a large animal model for phenylketonuria.

Hollow fiber enzyme-reactors with immobilized phenylalanine ammonia-lyase (PAL) were developed for the in vivo depletion of phenylalanine (Phe) in circulating blood. A series of experiments was conducted with a large animal model in order to explore its safety for clinical use. The level of red blood cells, white blood cells and platelets did not change during a 2-hr application of the reactors in anesthetized, heparinized dogs and monkeys with experimental hyperphenylalaninemia. No increase in blood urea nitrogen was observed due to generation of ammonia from PAL-catalyzed Phe breakdown. The other metabolic product, trans-cinnamic acid, was reported to be nontoxic. Repeated application of the PAL-reactors to the same animals did not produce untoward physiological or immunological reactions. These data suggest that PAL-reactors may be safe for in vivo use to control excess Phe brought about by fever, infection or pregnancy in phenylketonuric individuals otherwise balanced by a Phe-poor diet. Application of PAL-reactors may serve as a model for extracorporeal enzyme replacement in enzyme-deficiency diseases.

Depletion of phenylalanine in the blood of phenylketonuric patients using a PAL-enzyme reactor. An in vitro study.

Blood samples obtained from eight adult phenylketonuric individuals had a mean phenylalanine level of 25 mg/dl. When these samples were circulated through multitubular enzyme-reactors prepared with immobilized phenylalanine ammonia lyase an average of 77% of phenylalanine was metabolized within 30 minutes. We conclude that phenylalanine in human phenylketonuric blood is just as susceptible to metabolism by PAL-enzyme reactors as phenylalanine that is added to normal blood, or that is circulating in dogs and monkeys made hyperphenylalaninemic by experimental means.

Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria.

Multitubular enzyme reactors with immobilized enzymes were developed to achieve depletion of circulating substrate by extracorporeal means. To act as prototypes, reactors were prepared with immobilized L-phenylalanine ammonia-lyase, an enzyme that metabolizes phenylalanine to trans-cinnamic acid and ammonia without the need for a coenzyme. We report the first application of phenylalanine ammonia-lyase reactors in an extracorporeal circulation system in a patient with phenylketonuria. A phenylalanine level of 1.82 mmol/L (for the last 6 years) decreased to 1.24 mmol/L after 5.5. hours of treatment, without the enzyme entering the circulation. Total phenylalanine depletion from blood and tissue stores was estimated at 1800 mg. The hemodialysis-like procedure proved to be without side effects, specific for phenylalanine, and suitable in the management of pregnant women with phenylketonuria and late-onset hyperphenylalaninemia. The extracorporeal use of enzyme reactors for temporary enzyme replacement represents a new, safe, and effective therapeutic modality.